CN105713079B - Protein and its relevant biological material are improving the application in plant products - Google Patents
Protein and its relevant biological material are improving the application in plant products Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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Abstract
The invention discloses protein and its relevant biological material to improve the application in plant products.In application disclosed by the invention, protein is following A1), A2) or A3): A1) amino acid sequence is the protein of sequence 2；A2) by amino acid sequence shown in sequence 2 in sequence table by the substitution and/or deletion and/or addition of one or several amino acid residues and protein relevant to plant products；A3) in A1) or the obtained fused protein of N-terminal A2) or/and C-terminal connection label；Improving plant products includes improvement seed yield-related traits, and seed yield-related traits include at least one of wide spike length, grain number per spike, mass of 1000 kernel, grain length, grain, Primary branch number and Secondary branch number.It is demonstrated experimentally that wide, Primary branch number and Secondary branch number plant products can be improved by the spike length, grain number per spike, mass of 1000 kernel, grain length, the grain that improve plant using protein of the invention and its encoding gene.
Description
Technical field
The present invention relates to protein in field of biotechnology and its relevant biological material to improve the application in plant products.
Background technique
The production yields of grain is not only related to daily life, also restricts the economy of country to a certain extent
Situation.The country most as world population, China, the yield for improving grain are particularly important.In the past few decades, grain
Food yield increases substantially, and other than the improvement of tillage and cultivation management condition, the genetic improvement of crop varieties increases grain
Production has played important function.However, the raising of grain yield reaches bottleneck period in recent years.Currently, arable area gradually subtracts
It is few, and current improved variety yield potential is limited, and the world is faced with the challenge of new round grain security, it is therefore desirable to develop
New varieties with bigger yield potential.
Therefore, using Modern Molecular Biotechnology, the relevant key gene of yield is excavated, grain is improved by genetic engineering
Yield, for ensureing that national food production safety is of great significance.
Summary of the invention
The technical problem to be solved by the present invention is to how by improve spike length, grain number per spike, mass of 1000 kernel, grain length, grain it is wide, one
Secondary branch obstructs number and/or Secondary branch number to improve plant products.
In order to solve the above technical problems, present invention firstly provides protein following 1) -3) in it is any in application:
1) seed yield-related traits are improved；
2) preparation improvement seed yield-related traits product；
3) seed yield-related traits are cultivated and improves plant；
The seed yield-related traits include wide spike length, grain number per spike, mass of 1000 kernel, grain length, grain, Primary branch number and secondary
At least one of branch stalk number；
The entitled TaSPL20-7D of the protein is following A1), A2) or A3):
A1) amino acid sequence is the protein of sequence 2；
A2) by amino acid sequence shown in sequence 2 in sequence table by one or several amino acid residues substitution and/or
Deletion and/or addition and protein relevant to plant products；
A3) in A1) or the obtained fused protein of N-terminal A2) or/and C-terminal connection label.
Wherein, as once branch stalk and Secondary branch (such as rice), the seed yield-related traits are fringe to the plant
Length, grain number per spike, mass of 1000 kernel, grain length, grain be wide, at least one of Primary branch number and Secondary branch number；As the plant none
Secondary branch stalk and Secondary branch (such as wheat), the seed yield-related traits are that spike length, grain number per spike, mass of 1000 kernel, grain length and grain are wide
At least one of.
Wherein, sequence 2 is made of 388 amino acid residues.
In order to make A1) in protein convenient for purifying, amino acid sequence shown in sequence 2 can be formed in by sequence table
The amino terminal or carboxyl terminal of protein connect upper label as shown in Table 1.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned A2) in TaSPL20-7D protein, the substitution and/or missing of one or several amino acid residues
And/or it is added to substitution and/or deletion and/or addition no more than 10 amino acid residues.
Above-mentioned A2) in TaSPL20-7D protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biology
Expression obtains.
Above-mentioned A2) in the encoding gene of TaSPL20-7D protein can be by will be shown in 213-1379 of sequence 1
DNA sequence dna in lack the codons of one or several amino acid residues, and/or carry out one or several base-pairs missense it is prominent
Become, and/or is obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect label shown in table 1.
In order to solve the above technical problems, the present invention also provides biomaterial relevant to TaSPL20-7D it is described 1)-
3) in it is any in application, the biomaterial is following B1) any one of to B7):
B1 the nucleic acid molecules of TaSPL20-7D) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules or contain B2) recombinant vector of the expression cassette；
B4) contain B1) recombinant microorganisms of the nucleic acid molecules or contain B2) recombinant microorganism of the expression cassette or
Contain B3) recombinant microorganism of the recombinant vector；
B5) contain B1) the transgenic plant cells systems of the nucleic acid molecules or contain B2) transgenosis of the expression cassette
Plant cell；
B6) contain B1) Transgenic plant tissues of the nucleic acid molecules or contain B2) transgenosis of the expression cassette plants
Object tissue；
B7) contain B1) the genetically modified plants organs of the nucleic acid molecules or contain B2) transgenosis of the expression cassette plants
Sundries official.
In above-mentioned application, B1) nucleic acid molecules can be following b1)-b4) and in it is any:
B1) its coded sequence is the cDNA molecule or DNA molecular of 213-1379 nucleotide of sequence 1 in sequence table；
B2) nucleotide sequence is the DNA molecular cDNA molecule or DNA molecular of sequence 1 in sequence table；
B3) and b1) or b2) nucleotide sequence that limits has 75% or 75% or more identity, and encodes TaSPL20-
The cDNA molecule or genomic DNA molecule of 7D；
B4) the nucleotide sequence hybridization limited under strict conditions with b1) or b2), and encode the cDNA of TaSPL20-7D
Molecule or genomic DNA molecule.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also
To be RNA, such as mRNA or hnRNA.
Wherein, sequence 1 is made of 1445 nucleotide, and wherein DNA shown in 213-1379 nucleotide of sequence 1 points
Protein shown in sub- coded sequence 2.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation
Method is mutated the nucleotide sequence of coding TaSPL20-7D of the invention.Those have and this hair by manually modified
The nucleotide sequence 75% of bright isolated TaSPL20-7D or the nucleotide of higher identity, as long as coding TaSPL20-
7D and have the function of TaSPL20-7D, is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair
Amino acid sequence shown in bright coded sequence 2 composition protein nucleotide sequence have 75% or higher or 85% or
Higher or 90% or higher or 95% or higher identity nucleotide sequence.Identity can with the naked eye or computer software
It is evaluated.Using computer software, identity between two or more sequences can be indicated with percentage (%), can be with
For evaluating the identity between correlated series.
In above-mentioned application, the stringent condition is to hybridize at 68 DEG C in 2 × SSC, the solution of 0.1%SDS and wash film
2 times, each 5min, but in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and washes film 2 times, each 15min；
Or, hybridizing under the conditions of 65 DEG C in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS and washing film.
Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.
In above-mentioned application, B2) described in the nucleic acid molecules containing coding TaSPL20-7D expression cassette (TaSPL20-7D base
Because of expression cassette), it is the DNA for referring to express TaSPL20-7D in host cell, which not only may include starting TaSPL20-
The promoter of 7D genetic transcription may also include the terminator for terminating TaSPL20-7D genetic transcription.Further, the expression cassette is also
It may include enhancer sequence.Promoter for use in the present invention includes but is not limited to: constitutive promoter, tissue, organ and hair
Educate special promoter and inducible promoter.The example of promoter includes but is not limited to: the composing type of cauliflower mosaic virus
Promoter 35S: the wound-inducible promoter from tomato, leucine aminopeptidase (" LAP ", Chao et al. (1999)
Plant Physiol 120:979-992)；Chemical inducible promoter from tobacco, pathogenesis correlation 1 (PR1) is (by water
Poplar acid and BTH (diazosulfide -7- carbothioic acid S-methyl ester) induction)；Tomato protease inhibitors II promoter (PIN2)
Or LAP promoter (available methyl jasmonate induction)；Heat-shock promoters (United States Patent (USP) 5,187,267)；Tetracycline lures
Conductivity type promoter (United States Patent (USP) 5,057,422)；Seed specific promoters, such as Millet Seed specificity promoter pF128
(CN101063139B (Chinese patent 200710099169.7)), the special promoter of seed storage protein matter is (for example, Kidney bean ball
(Beachy et al. (1985) EMBO is J.4:3047- for the promoter of albumen, napin, oleosin and soybean beta conglycin
3053)).They can be used alone or are used in combination with other plant promoters.All references cited herein is complete
Text reference.Suitable transcription terminator includes but is not limited to: Agrobacterium nopaline syntase terminator (NOS terminator), flower coconut palm
Cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase
Terminator (see, e.g.: Odell et al. (I985)Nature313:810；Rosenberg et al. (1987) Gene, 56:125；
Guerineau et al. (1991) Mol.Gen.Genet, 262:141；Proudfoot(1991)Cell,64:671；Sanfacon
Et al. Genes Dev., 5:141；Mogen et al. (1990) Plant Cell, 2:1261；Munroe et al. (1990) Gene,
91:151；Ballad et al. (1989) Nucleic Acids Res.17:7891；Joshi et al. (1987) Nucleic Acid
Res.,15:9627)。
The recombinant vector of the TaSPL20-7D expression casette can be contained with existing expression vector establishment.The plant
Expression vector includes double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment.As pAHC25, pBin438,
PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or
PCAMBIA1391-Xb (CAMBIA company) etc..The plant expression vector also may include 3 ' end non-translational regions of foreign gene
Domain, i.e., comprising polyadenylation signals and any other DNA fragmentation for participating in mRNA processing or gene expression.The polyadenylic acid letter
Number bootable polyadenylic acid is added to 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces (Ti) plasmid gene (such as nopaline
Synthase gene Nos), plant gene (such as soybean storage protein genes) 3 ' end transcription non-translational region all have similar functions.
When using gene constructed plant expression vector of the invention, enhancer, including translational enhancer or transcriptional enhancer also can be used,
These enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must read with coded sequence
Frame is identical, to guarantee the correct translation of entire sequence.The source of the translation control signal and initiation codon be it is extensive,
Can be it is natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or structural gene.In order to just
In transgenic plant cells or plant are identified and screened, plant expression vector used can be processed, it can as being added
The coding expressed in plant can produce the enzyme of color change or gene (gus gene, luciferase genes of luminophor
Deng), the marker gene of antibiotic (if assigned the nptII gene to kanamycins and associated antibiotic resistance, assigns to herbicide
The bar gene of phosphinothricin resistance assigns the hph gene to antibiotic hygromycin resistance, and assigns to methotrexate resistance
Dhfr gene is assigned to the EPSPS gene of glyphosate) or (such as anti-herbicide base such as anti-chemical reagent marker gene
Cause), provide metabolism mannose ability mannose-6-phosphate isomerase gene.It, can not from the security consideration of genetically modified plants
Add any selected marker, transformed plant is directly screened with adverse circumstance.
In above-mentioned application, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.The plasmid is concretely
PMD18-T or pAHC25.
B3) for encoding the DNA of TaSPL20-7D shown in 213-1379 containing sequence 1 of the recombinant vector
Sequence；Further B3) recombinant vector concretely pCUbi 1390-TaSPL20-7D.The pCUbi 1390-
TaSPL20-7D is the 213- that the DNA sequence dna between Kpn I and Spe the I recognition site of pCUbi 1390 is replaced with to sequence 1
DNA sequence dna shown in 1379 keeps other DNA sequence dnas constant, obtains the recombinant vector of expression TaSPL20-7D.
In above-mentioned application, the microorganism can be yeast, bacterium, algae or fungi.Wherein, bacterium may be from Escherichia
(Escherichia), Erwinia (Erwinia), Agrobacterium tumefaciems category (Agrobacterium), Flavobacterium
(Flavobacterium), Alcaligenes (Alcaligenes), pseudomonas (Pseudomonas), Bacillus
(Bacillus) etc..
In above-mentioned application, the transgenic plant cells system, Transgenic plant tissue and genetically modified plants organ are not wrapped
Include propagation material.
In order to solve the above technical problems, the present invention also provides improve plant products product；The product contains
TaSPL20-7D or the biomaterial.
In the said goods, the raising plant products product can also be incited somebody to action using TaSPL20-7D as active constituent
The composition that TaSPL20-7D and other raising plant products substances are combined is as active constituent.
In order to solve the above technical problems, the present invention also provides a kind of method for improveing seed yield-related traits, it is described
Method includes that the encoding gene of from the importing TaSPL20-7D to recipient plant obtains genetically modified plants；With the recipient plant phase
, Primary branch number wider than, spike length, grain number per spike, mass of 1000 kernel, grain length, the grain of the genetically modified plants and/or Secondary branch number increase
Add.
In order to solve the above technical problems, the present invention also provides a kind of method for cultivating output increased plant, the method
Encoding gene including importing from TaSPL20-7D to recipient plant obtains genetically modified plants；Compared with the recipient plant, institute
The yield for stating genetically modified plants increases.
In the method for above-mentioned improvement seed yield-related traits and the method for cultivation output increased plant, the TaSPL20-
The coded sequence of the encoding gene of 7D can be 213-1379 DNA moleculars of sequence 1 in sequence table.
In the method for above-mentioned improvement seed yield-related traits and the method for cultivation output increased plant, the recipient plant
It can be monocotyledon or dicotyledon.The monocotyledon concretely gramineae plant, such as rice or wheat.
In an embodiment of the present invention, encoding gene (the i.e. 213-1379 nucleosides of sequence 1 of the TaSPL20-7D
DNA molecular shown in acid) it is imported by the TaSPL20-7D gene recombinant vectors containing TaSPL20-7D expression casette
In purpose plant.
In the method for above-mentioned improvement seed yield-related traits and the method for cultivation output increased plant, wherein described
TaSPL20-7D gene can be modified first as follows, then import in receptor seed plant, to reach better expression effect:
1) it modifies and optimizes according to actual needs, so that gene efficient expression；For example, can be according to recipient plant institute partially
The codon of love changes its codon while keeping the amino acid sequence of TaSPL20-7D gene of the present invention to meet
Plant-preference；In optimization process, it is desirable that certain G/C content is kept in the coded sequence after optimization, to be best implemented with
The high level expression of quiding gene in plant, wherein G/C content can be 35%, be more than 45%, more than 50% or more than about 60%；
2) gene order of neighbouring initial methionine is modified, so that translation effectively starting；For example, using in plant
The effective sequence known is modified；
3) it is connect with the promoter of various plants expression, in favor of its expression in plant；The promoter may include
Composing type, induction type, timing adjusting, growth adjustment, Chemical Regulation, tissue are preferably and tissue-specific promoter；Promoter
Selection will need with expression time and space and be changed, and also depend on target kind；Such as the specificity of tissue or organ
Promoter is expressed, receptor as needed is depending on what period of development；Although demonstrating many from dicotyledon
Promoter can act in monocotyledon, and vice versa, but it is desirable to select dicot promoters are used for
Expression in dicotyledon, monocotyledonous promoter is for the expression in monocotyledon；
4) it is connect with suitable transcription terminator, can also be improved the expression efficiency of gene of the present invention；Such as from
The tml of CaMV, from the E9 of rbcS；Any known available terminator to work in plant can be with the present invention
Gene is attached；
5) enhancer sequence is introduced, such as intron sequences (such as from Adhl and bronzel) and viral leader sequence
(such as from TMV, MCMV and AMV).
The TaSPL20-7D expression vector can by using Ti-plasmids, Ri plasmid, plant viral vector, directly
The conventional biology methods such as DNA conversion, microinjection, conductance, mediated by agriculture bacillus, particle gun convert plant cell or tissue, and
The plant tissue of conversion is cultivated into plant.
The method also includes the screening expression institutes from the plant of encoding gene for importing TaSPL20-7D shown in sequence 2
The plant for stating encoding gene obtains the transgenic wheat.
In the present invention, the yield can be the yield of seed.
In the present invention, the genetically modified plants are interpreted as not only including by the TaSPL20-7D genetic transformation purpose plant
Obtained first generation genetically modified plants also include its filial generation.For genetically modified plants, the gene can be bred in the species,
The gene transfer can also be entered to other kinds of same species with traditional breeding techniques, particularly including in commercial variety.It is described
Genetically modified plants include seed, callus, intact plant and cell.
In the present invention, the plant can be monocotyledon or dicotyledon.The monocotyledon concretely standing grain
Graminaceous plant, such as rice or wheat.
It is demonstrated experimentally that the spike length of plant, grain number per spike, thousand can be improved in TaSPL20-7D and its encoding gene of the invention
Weight, grain length, grain be wide, Primary branch number and Secondary branch number: the extremely significant height of spike length of three Ge Zhuan TaSPL20-7D rice strains
In turning empty carrier rice, 8.0%, 15.2% and 9.6% is higher by than turning empty carrier rice respectively；Three turn TaSPL20-7D rice
The grain number per spike of strain is extremely significant higher than empty carrier rice is turned, and is higher by 33.9%, 53.9% and than turning empty carrier rice respectively
31.5%；The Primary branch number of three Ge Zhuan TaSPL20-7D rice strains is extremely significant higher than empty carrier rice is turned, respectively than turning
Empty carrier rice is higher by 45.2%, 35.5% and 27.4%；The Secondary branch number of three Ge Zhuan TaSPL20-7D rice strains is aobvious
It writes to be higher than and turns empty carrier rice, be higher by 23.6%, 48.6% and 31.9% than turning empty carrier rice respectively；Three turn TaSPL20-
The mass of 1000 kernel of 7D rice strain, which is all remarkably higher than, turns empty carrier rice, is higher by 9.4%, 5.3% and than turning empty carrier rice respectively
1.9%；The grain length of three Ge Zhuan TaSPL20-7D rice strains is extremely significant higher than empty carrier rice is turned, respectively than turning empty carrier water
Rice is higher by 6.0%, 5.5% and 2.8%；The grain of three Ge Zhuan TaSPL20-7D rice strains it is wide it is extremely significant be higher than turn empty carrier water
Rice is higher by 2.6%, 1.7% and 2.0% than turning empty carrier rice respectively.Show in actual production, utilization can be passed through
Spike length, grain number per spike, mass of 1000 kernel, grain length, the grain that TaSPL20-7D and its encoding gene improve plant be wide, Primary branch number and secondary
Branch obstructs number to improve plant products.
Detailed description of the invention
Fig. 1 is the relative expression quantity for being overexpressed three strains of TaSPL20-7D transgenic paddy rice.Wherein, L1-L3 divides table table
Show that TaSPL20-7D-A, TaSPL20-7D-B and TaSPL20-7D-C, WT indicate that rice KitaaKe, Vector indicate pCUbi
1390。
Fig. 2 is relative expression quantity of the TaSPL20-7D in wheat different development stage different tissues；A and B is respectively half
The relative expression's situation of quantitative PCR and Real-time PCR Analysis TaSPL20-7D in different times different tissues.
Fig. 3 is relative expression quantity of the different development stage TaSPL20-7D in each organ of wheat fringe portion；A and B are respectively
Fringe develops the relative expression quantity of early period and fringe development later stage TaSPL20-7D in each organ of wheat fringe portion
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Wheat (Triticum aestivum L.) kind Yanzhan4110 in following embodiments: weak spring habit, it is precocious, it is mature
Phase is identical as control Henan wheat No. 18.Seedling is upright, and tillering ability is strong, and leaf color is dark green, and blade width is short.Plant height 75cm, plant type is compacter,
It is rushed on boot leaf, lodging resistance is preferable.Wheat (Triticum aestivum L.) kind Yanzhan4110 (Zhao Tianxiang etc., EMS mutagenesis six
The morphological mutant identification of times body wheat Yanzhan4110 and analysis Scientia Agricultura Sinica, 2009,42 (3): 755-764) public can
It is obtained from applicant, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes and uses.Come
Material is saved derived from laboratory, the public can obtain from applicant, be only limitted to for repeating the present invention.
In following embodiments carrier pCUBi 1390 (the degeneration-resistant functional analysis Acta Agronomica Sinica of paddy gene OsASIE1,
2011 37 (10) 1771-1778.) public can obtain from applicant, the biomaterial only attach most importance to duplicate invention correlation it is real
Used in testing, it not can be used as other purposes and use.
Agrobacterium tumefaciems EHA105 (Agrobacterium tumefaciens Strain in following embodiments
EHA105): Biovector Science Lab, Inc product.
In following embodiments rice KitaaKe (rice breeding of high photosynthetic efficiency progress, Biotechnological Advances, 2014 4
(3) 153-157.) public can obtain from applicant, and which only attaches most importance to used in the related experiment of duplicate invention, can not
It is used as other purposes.
The functional verification of embodiment 1, TaSPL20-7D gene
Wheat (Triticum aestivum L.) kind Yanzhan4110 seedling total serum IgE is extracted with TRIZOL, it is anti-with M-MLV
Transcript reagent box synthesizes the first chain cDNA (Invitrogen), and using this cDNA as template, primers F 1 and R1 carry out PCR amplification, obtains
To the pcr amplification product of 1445bp.
1-20 of F1:5'-AAGAGTTATTGCCAACGGAC-3'(sequence 1)；
1423-1445 reverse complementary sequences of R1:5'-CTAATGTGGTGTTCTAACTGTGG-3'(sequence 1).
By sequencing, the sequence of the PCR product is as shown in sequence 1 in sequence table, 213-1379 nucleotide of sequence 1
For code area, unnamed gene shown in the 213-1379 nucleotide by sequence 1 is TaSPL20-7D gene, which is compiled
The protein of code is named as TaSPL20-7D, and amino acid sequence is sequence 2 in sequence table.
One, the acquisition of transgenic paddy rice
1, the acquisition of recombinant vector
Kpn I and the Spe I of carrier pCUbi 1390 is identified that the DNA fragmentation between sequence replaces with the 213- of sequence 1
Gene shown in 1379 nucleotide (TaSPL20-7D gene), keeps the other sequences of carrier constant, obtains recombinant vector, will
The recombinant vector is named as pCUbi 1390-TaSPL20-7D.TaSPL20-7D gene in pCUbi 1390-TaSPL20-7D
Expression is started by maize ubiquitin promoter.
PCUbi 1390-TaSPL20-7D is imported in Agrobacterium tumefaciems EHA105, recombinant bacterium is obtained, which is ordered
Entitled E-pCUbi 1390-TaSPL20-7D；Carrier pCUbi 1390 is imported in Agrobacterium tumefaciems EHA105, is recombinated
The recombinant bacterium is named as E-pCUbi 1390 by bacterium.
2, the acquisition of transgenic paddy rice
Using rice KitaaKe as receptor, E-pCUbi 1390-TaSPL20-7D is utilized by agrobacterium mediation converted method
The mature embryo callus of rice transformation KitaaKe is taken root, strong sprout process, most by the screening and differentiation of resistant calli
32 T are obtained eventually0In generation, turns TaSPL20-7D trans-genetic hybrid rice strain.
Specific method for transformation is according to document (Hiei Y, Ohta S, Komari T, Kumashiro T.Efficient
transformation of rice(Oryza saliva L.)mediated by Agrobacterium and sequence
Analysis of boundaries of the T-DNA.Plant J, 1994,6 (2): 271-282.) in mediated by agriculture bacillus
Plantlet Regeneration in Mature Embryo Culture of Rice Transformation of Callus method carry out.
According to the Plantlet Regeneration in Mature Embryo Culture of Rice Transformation of Callus method of above-mentioned mediated by agriculture bacillus, with 1390 rice transformation of E-pCUbi
KitaaKe obtains T0In generation, turns empty carrier rice.
3, the Molecular Identification of transgenic paddy rice
Extract T0In generation, turns the genomic DNA of TaSPL20-7D rice strain, with AS2-F1 (5 '-
GGCGAGCGACGACTTCC-3 ') and AS2-R1 (5 '-TGTCGTTGTTCTGGTCGGAG-3 ') carry out PCR amplification, identification obtains
23 positive T0In generation, turns TaSPL20-7D rice strain.
By positive T0In generation, turns TaSPL20-7D rice strain and is selfed, and obtains T1In generation, turns TaSPL20-7D rice paddy seed；It will
T1In generation, turns the sowing of TaSPL20-7D rice paddy seed, and carries out resistance screening with 1 ‰ hygromycin aqueous solutions, obtains T1In generation, turns
TaSPL20-7D plant is cultivated to obtain T under normal operation2In generation, turns TaSPL20-7D rice paddy seed；By T2In generation, turns
The sowing of TaSPL20-7D rice paddy seed, and resistance screening is carried out with 1 ‰ hygromycin aqueous solutions, obtain T3In generation, turns TaSPL20-7D plant
Strain, chooses three T3Generation turn TaSPL20-7D strain carry out phenotypic evaluation, be named as TaSPL20-7D-A, TaSPL20-7D-B and
TaSPL20-7D-C。
Using expression of the target gene on rna level in each plant of Semi quantitative PCR analysis, as a result as shown in Figure 1.
Use the rice Ubiqutin gene of constitutive expression as internal reference, the primer sequence of design is as follows:
The primer sequence for detecting gene TaSPL20-7D gene expression is as follows:
TaSPL20-RT-F:5 '-CGGAGAATGACGACCAC-3 '；
TaSPL20-RT-R:5 '-GTCGTTGTTCTGGTCGG-3 '；
The primer sequence for detecting rice Ubiqutin gene expression is as follows:
OsUBQ5-F:5 '-ACCACTTCGACCGCCACTACT-3 '；
OsUBQ5-R:5 '-ACGCCTAAGCCTGCTGGTT-3 '.
The results show that the target gene imported in transgenic plant is expressed.
Extract T0In generation, turns the genomic DNA of empty carrier rice, with hygromycin gene primer HYG-F (5 '-
CTTCTGCGGGCGATTTGT-3 ') and HYG-R (5 '-CAGCGTCTCCGACCTGAT-3 ') progress PCR amplification, it identifies and obtains sun
Property T0In generation, turns empty carrier rice.
By positive T0In generation, turns empty carrier rice and is selfed, and carries out resistance screening with 1 ‰ hygromycin aqueous solutions, until
To T3Turn empty carrier rice for homozygosis.
4, transgenic paddy rice phenotypic evaluation
Fetch water rice KitaaKe (wild type control, WT), T3Turn empty carrier rice (Vector) and T for homozygosis3In generation, turns
The seed of TaSPL20-7D rice strain (TaSPL20-7D-A, TaSPL20-7D-B and TaSPL20-7D-C), after seedling, big
Maturation is grown in field.At least 30 plants of each strain, experiment is repeated 3 times.
After transgenic paddy rice full maturity, to wild type, turns empty carrier rice and turn the progress of TaSPL20-7D rice strain
Phenotypic evaluation, respectively to the spike length of each rice, grain number per spike, mass of 1000 kernel, grain length, grain be wide, Primary branch number and Secondary branch number into
Row statistical analysis.Statistical result such as table 2.
Table 2, transgenic paddy rice maturity period phenotype statistical result
| Character | WT | Turn empty carrier plant | TaSPL20-7D-A strain | TaSPL20-7D-B strain | TaSPL20-7D-C strain |
| Spike length | 12.5±0.9B | 12.5±1.1B | 13.5±1.3A | 14.4±1.4A | 13.7±1.1A |
| Grain number per spike | 53.3±8.3B | 50.5±9.2B | 67.6±10.8A | 77.7±14.1A | 66.4±8.0A |
| Primary branch number | 6.6±0.7B | 6.2±0.5B | 9.0±1.3A | 8.4±1.2A | 7.9±0.7A |
| Secondary branch number | 7.7±1.9b | 7.2±1.9b | 8.9±2.4a | 10.7±2.9a | 9.5±2.2a |
| Mass of 1000 kernel | 26.7±0.5b | 26.6±0.8b | 29.1±0.9a | 28.0±0.8a | 27.1±0.6a |
| Grain length | 7.52±0.08B | 7.46±0.08B | 7.91±0.17A | 7.87±0.17A | 7.67±0.10A |
| Grain is wide | 3.52±0.06B | 3.55±0.04B | 3.61±0.04A | 3.58±0.05A | 3.59±0.04A |
Annotation: in a line, in 0.01 level, there are poles to the corresponding character of the plant of mark B for the plant of mark capitalization A
Significant difference, in 0.05 level, there are significant differences to the corresponding character of the plant of mark b for the plant of mark lowercase a.
Spike length: wild rice and the basic indifference of the spike length for turning empty carrier rice, three turn TaSPL20-7D rice strain
The spike length of system is extremely significant higher than empty carrier rice is turned, and is higher by 8.0%, 15.2% and 9.6% than turning empty carrier rice respectively.
Grain number per spike: wild rice and the basic indifference of the grain number per spike for turning empty carrier rice, three turn TaSPL20-7D water
The grain number per spike of rice strain is extremely significant higher than empty carrier rice is turned, and is higher by 33.9%, 53.9% and than turning empty carrier rice respectively
31.5%.
Primary branch number: wild rice and turning this indifference of the Primary branch base of empty carrier rice, and three turn
The Primary branch number of TaSPL20-7D rice strain is extremely significant higher than empty carrier rice is turned, and is higher by respectively than turning empty carrier rice
45.2%, 35.5% and 27.4%.
Secondary branch number: wild rice and turning this indifference of the Secondary branch base of empty carrier rice, and three turn
The Secondary branch number of TaSPL20-7D rice strain, which is all remarkably higher than, turns empty carrier rice, is higher by respectively than turning empty carrier rice
23.6%, 48.6% and 31.9%.
Mass of 1000 kernel: wild rice and the basic indifference of the mass of 1000 kernel for turning empty carrier rice, three turn TaSPL20-7D water
The mass of 1000 kernel of rice strain, which is all remarkably higher than, turns empty carrier rice, is higher by 9.4%, 5.3% and than turning empty carrier rice respectively
1.9%.
Grain length: wild rice and the basic indifference of the grain length for turning empty carrier rice, three turn TaSPL20-7D rice strain
The grain length of system is extremely significant higher than empty carrier rice is turned, and is higher by 6.0%, 5.5% and 2.8% than turning empty carrier rice respectively.
Grain is wide: wild rice and the wide basic indifference of the grain for turning empty carrier rice, three turn TaSPL20-7D rice strain
The grain of system is wide extremely significant higher than empty carrier rice is turned, and is higher by 2.6%, 1.7% and 2.0% than turning empty carrier rice respectively.
The experimental results showed that TaSPL20-7D can dramatically increase spike length of rice, branch of the ear of grain and grain number per spike, while increasing grain length
With grain is wide and mass of 1000 kernel.
The expression of embodiment 2, TaSPL20-7D gene in different tissues
One, expression of the TaSPL20-7D gene in different tissues
The different tissues position is taken to be in wheat (Triticum aestivum L.) kind Yanzhan4110 growth different times
Sample, through liquid nitrogen frozen, -80 DEG C are saved backup.Obtaining specimen material includes: seedling stage root (SR), seedling stage apical meristem
(SC), 4 leaf phase apical meristem (4C), 6 leaf phase apical meristems (6C), period of seedling establishment apical meristem (GC)), seedling stage
Leaf (SL), root (BR) after booting, section (BN), internode (BI), leaf sheath (BS), leaf (BL), fringe (BE) (0.5cm fringe (0.5E),
2cm fringe (2E), 3cm fringe (3E), 6cm fringe (6E), 8cm fringe (8E), 10cm fringe (10E)), the fringe (BE) in booting later period, florescence
Fringe (FE), 21 days 4 days seeds (4G) of Hua Hou, 7 days seeds (7G) of Hua Hou, Hua Hou 14 days seed (14G) He Huahou seeds
(21G)。
The total serum IgE for extracting above-mentioned Liquid nitrogen storage sample respectively with TRIZOL is detected using real-time quantitative and semi-quantitative method
Differential expression of the TaSPL20-7D gene in different times different tissues.Use the Actin gene of constitutive expression as internal reference,
The primer sequence of design is as follows:
The primer sequence for detecting gene TaSPL20-7D gene expression is as follows:
TaSPL20-RT-F:5 '-CGGAGAATGACGACCAC-3 '；
TaSPL20-RT-R:5 '-GTCGTTGTTCTGGTCGG-3 '；
The primer sequence for detecting Gene A ctin expression is as follows:
Actin-F:5 '-CTCCCTCACAACAACAACCGC-3 '；
Actin-R:5 '-TACCAGGAACTTCCATACCAAC-3 '.
Real-time quantitative and sxemiquantitative experimental result are as shown in Fig. 2, expression quantity of the TaSPL20-7D gene on young fringe is obvious
Higher than other tissues.
Two, spatial and temporal expression situation of the TaSPL20-7D gene in fringe portion
Each organ of wheat (Triticum aestivum L.) kind Yanzhan4110 fringe portion different development stage is taken
Sample, through liquid nitrogen frozen, -80 o C are saved backup.The total serum IgE for extracting above-mentioned Liquid nitrogen storage sample respectively with TRIZOL, using real-time
Quantitative approach detects TaSPL20-7D from the young fringe of 3cm to fringe when blooming in the table of glumelle, lemma, gynoecium, stamen and cob
Up to situation.It uses the Actin gene of constitutive expression as internal reference, detects TaSPL20-7D gene primer and detection Gene A ctin
The same step 1 of the primer of expression.
Real-time quantitative experimental result is as shown in figure 3, TaSPL20-7D gene is a large amount of in the fringe development glumelle of early period, lemma
Expression.
Claims (6)
1. protein is following 1) -3) in it is any in application:
1) seed yield-related traits are improved；
2) preparation improvement seed yield-related traits product；
3) seed yield-related traits are cultivated and improves plant；
The seed yield-related traits include wide grain number per spike, mass of 1000 kernel, grain length, grain, in Primary branch number and Secondary branch number
At least one；
The protein is the protein that amino acid sequence is sequence 2；
The plant is monocotyledon；The monocotyledon is gramineae plant.
2. biomaterial relevant to protein described in claim 1 in claim 1 described 1) -3) in it is any in answer
With the biomaterial is any one of following B1) to B4):
B1 the nucleic acid molecules of protein described in claim 1) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules or contain B2) recombinant vector of the expression cassette；
B4) contain B1) recombinant microorganisms of the nucleic acid molecules or contain B2) recombinant microorganism of the expression cassette or contain
B3) the recombinant microorganism of the recombinant vector.
3. applying according to claim 2, it is characterised in that: B1) nucleic acid molecules are following b1) or b2):
B1) its coded sequence is the cDNA molecule or DNA molecular of 213-1379 nucleotide of sequence 1 in sequence table；
B2) nucleotide sequence is the DNA molecular cDNA molecule or DNA molecular of sequence 1 in sequence table.
4. improving plant products product, it is characterised in that: the product contains protein described in claim 1 or right is wanted
Seek biomaterial described in 2 or 3.
5. a kind of method for improveing seed yield-related traits, including protein described in claim 1 is imported into recipient plant
Encoding gene obtain genetically modified plants；Compared with the recipient plant, grain number per spike, mass of 1000 kernel, the grain of the genetically modified plants
It grows, grain is wide, Primary branch number and/or Secondary branch number increase；The recipient plant is monocotyledon or dicotyledon；Institute
Stating monocotyledon is gramineae plant.
6. according to the method described in claim 5, it is characterized by: the coding of the encoding gene of protein described in claim 1
Sequence is 213-1379 DNA moleculars of sequence 1 in sequence table.
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| CN109912702B (en) * | 2017-12-13 | 2021-03-16 | 中国科学院遗传与发育生物学研究所 | Application of protein OsARE1 in regulation and control of low nitrogen resistance of plants |
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| Title |
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| 小麦产量相关基因TaSPLs的克隆和功能分析;刘霞;《中国优秀硕士学位论文全文数据库》;20160115(第1期);第3页倒数第2段，第12页最后一段，第24页最后一段，第31页2.5节 * |
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